Method to Enhance an Immune Response of Nucleic Acid Vaccination

ABSTRACT

A composition comprising liposomes associated with a nucleic acid operatively encoding an antigenic protein and with an assistor protein, wherein the assistor protein shares at least one epitope with the antigenic protein, and wherein the nucleic acid and said assistor protein are associated with the same liposomes is described. The composition provides an improved immune response compared to mixtures of liposomes some of which are associated with the nucleic acid and some of which are associated with the assistor protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. Nos.14/447,556 which is a divisional of 13/292,778 filed 9 Nov. 2011, whichis a continuation of U.S. application Ser. No. 12/566,587 filed 24 Sep.2009, now U.S. Pat. No. 8,075.896, issued on 13 Dec. 2011, which is acontinuation of U.S. application Ser. No. 10/520,169 filed 27 Apr. 2005,now U.S. Pat. No. 7,604,803, issued 20 Oct. 2009, which is a nationalstage application of PCT/GB03/02935 filed 7 Jul. 2003, which claimspriority to European Patent Application 02254733.5 filed 5 Jul. 2002.The contents of the above patent applications and patents areincorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention relates to compositions for the co-delivery ofnucleic acid and protein. Co-delivery means delivery to the same cell.The compositions are useful for generating an immune response. Inparticular, the nucleic acid operatively encodes an antigenic protein orprotein thereof, the sequence of which is homologous, preferablyidentical, to that of an ‘assistor protein’ which forms part of thecompositions.

Protein antigens from pathogens have long been used in vaccines,designed to elicit neutralising antibody or cell-mediated immuneresponses in the recipient, specific for the antigen. Proteins howeverare generally not good at eliciting certain types of cell-mediatedimmune response, particularly the generation of effector T-cells(including cytotoxic T-cells), which are a desirable component of theresponse for a great many vaccines (particularly those directed againstintracellular pathogens or cancer antigens). Latterly, vaccines havebeen developed based on naked DNA, usually plasmid DNA produced from Ecoli but containing appropriate promoter sequences for expression inmammalian cells. These latter vaccines have transpired to be good atgenerating cell mediated immunity (involving effector T-cells, such asinterferon-.gamma. secreting antigen-specific T-cells andantigen-specific cytotoxic T-cells), but are poor at generatingantibodies against the encoded and expressed antigen. Antibodies are animportant component of the protective immune response for a great manypathogens-particularly bacteria and certain viruses such as theinfluenza viruses. Various remedies have been proposed and explored torectify the deficiencies of DNA-based vaccines as described below.

Liposomal formulation has been used to enhance the immunogenicity ofvaccine antigens, its the protein form, for many years. Liposomalformulation has also teen applied in recent years to the formulation ofDNA for vaccine purposes. There are studies which have described theco-formulation of plasmid DNA with proteins using liposome. However,studies of liposomal co-formulation of DNA with protein have generallyused plasmids encoding immunostimulatory cytokines or other biologicallyactive proteins—other than antigen itself. To incorporate the proteinform of the antigen itself into a vaccine composition containing anucleic acid which is designed to express the protein in vivo would seemunnecessary. We are aware of only one publication which has used proteinantigen as an additive in the formulation alongside DNA(Alvarez-Lajonchere, L. et al., Mem Inst Oswaldo Cruz, Rio de Janiero,97(1):95-99, January 2002). Unlike the present invention, no enhancementof antibody response was seen by these authors in co-formulations of theantigen-encoding DNA and its cognate protein compared to immunisationswith the protein alone. The formulations used by Alvarez-Lajonchere, etal., comprised mixtures of the active nucleic acid (a plasmid encodingthe core antigen of hepatitis-C virus) plus irrelevant carrier DNA andpolyethylene glycol and the protein. Following injection, the proteinand the active DNA (which were not physically associated in the mixture)would diffuse independently and reach antigen presaging cellsseparately. The negative findings of Alvarez-Lajonchere would suggest,to a person skilled in the art, that formulation of protein with itscognate DNA was not a promising way to achieve improved immuneresponses, at least not improved antibody responses.

In WO-A-9938733 the immune response to a nucleic acid vaccine isproposed to be enhanced by simultaneous administration of a cognateprotein. The two components do not need to be administered in the samecomposition. Both must merely be administered during the induction phaseof the immune response with the protein preferably being masked or heldhack until after the nucleic acid has primed the immune system. In someexamples a vaccine comprised naked DNA and naked protein antigen inphysical admixture. In others the protein antigen was formulated fordelayed please in a biodegradable polymer-alum formulation admixed withnaked DNA.

In WO-A-9728818 vaccines are intended to deliver nucleic acid andprotein antigen into antigen presenting cells. The nucleic acid mayexpress the same protein as the protein antigen. The nucleic acid andprotein are complexed, e.g., by covalent conjugation. The complex may beformulated as a synthetic virus-like particle. It is also suggested thatliposomal systems may be used but there are no examples as to how bothprotein and nucleic acid should be incorporated into such systems, nordoes the specification include any quantitative results for in vivotests but predicts results which may not in practice occur, especiallyclass II responses.

It is known that non-coding plasmid DNA has an immuno-adjuvant actionwhen coentrapped with peptides in liposomal vesicles (Gursel, M., et al.Vaccine (1999) 17:1376-1383) and that DNA with CpG motifs has an immunoadjuvant effect on naked DNA and peptide vaccines (Klinman, D. M., etal., Vaccine (1999) 17:19-25).

BRIEF SUMMARY

In the present invention however, we imagined that if we contrived tophysically associate nucleic acid, such as DNA, together with itscognate protein and entrap them, that the two entities would arrive atantigen-presenting cells together, resulting in the processing andpresentation of the acquired protein form of the antigen, together withthe expression of the DNA-encoded form of the antigenic protein in thesame cell. Since antigen processing of expressed proteins occurs by adifferent pathway and with kinetics that are somewhat different to thatfor acquired proteins, we imagined that such co-delivery of DNAassociated with its cognate protein would provide an opportunity for anadditive or synergistic effect of these two modes of antigenpresentation, and an improved immune response. Now we have tested thisnew hypothesis with vesicular formulations of DNA and its cognateprotein which provide for the association of the DNA and protein. UnlikeAlvarez-Lajonchere, et al., we have found that special vaccinecompositions are possible, of DNA associated (via liposomes) with itscognate protein, called an ‘assistor protein’, wherein enhanced antibodyresponses are observed following immunisation (compared to immunisationwith protein alone, or with DNA alone). We find that if the DNA and theprotein are formulated in separate particles, and the particles aremixed, then we see no enhancement of antibody production. Theseobservations are consistent with our theory of co-delivery of DNA andthe assistor protein to the same antigen presenting cell, although weacknowledge that there may be other theoretical explanations that cannot be excluded at this time.

This invention provides a composition for the co-delivery to a cell of anucleic acid and an assistor protein comprising vesicles formed ofamphiphilic components, wherein the nucleic acid operatively encodes anantigenic protein or portion thereof which shares at least one epitopewith the assistor protein, the composition comprising said nucleic acidand said assistor protein being associated with the same vesicles as oneanother.

The term assistor protein refers to whole proteins or fragments ofproteins, proteins of a single type or proteins of different types.

The antigenic protein encoded by the nucleic acid is generally theprotein of interest, i.e., the target antigen against which a beneficialimmune response is desired in a subject. The assistor protein isgenerally identical to the expressed form of nucleic-acid encodedantigenic protein, i.e., the conjugate protein of the nucleic acid. Theantigenic protein and or the assistor protein may each (severally)comprise the full sequence of the naturally occurring protein from therelevant source. Preferably the nucleic acid encodes the entirenaturally occurring protein antigen. Alternatively, the nucleic acid mayencode a portion only of the natural protein, including at least one ofthe epitopes of the assistor protein. In one favourable embodiment ofthe invention, said epitope is a B-cell epitope which is exposed on thesurface of an infectious agent in it's naturally occurring form. Thenucleic acid may encode a portion only of the natural protein, includingat least one of the epitopes of the naturally occurring agent. The agentis, for instance, a microorganism, for instance a bacterium, or a yeastor a virus. Similarly, the assistor protein should contain epitopesderived from the respective source which are (in one embodiment) surfaceaccessible when the source is in its natural environment. Alternativelyand usefully, both antigenic protein and assistor protein shareepitope(s) with a secreted toxic product of a pathogen, such as tetanustoxin, appropriate to neutralisation of such toxin. Likewise, bothantigenic protein and assistor protein may share epitope(s) with eachother, which is/are also shared with a secreted product of a pathogenother than a toxin, such as the interleukin-10 analogue encoded byEpstein-Barr virus and secreted by cells infected with the Epstein-Burr

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows an assessment of the formulations (Table 2) for HAantigenicity by capture ELISA.

FIG. 2 shows measurement of anti influenza (major protein HA) antibodyresponse 16 days post one dose.

FIG. 3 shows measurement of anti influenza (major protein HA) antibodyresponse 28 days post one dose.

FIG. 4 shows measurement of anti influenza (major protein HA) antibodyresponse 15 days post second dose.

FIG. 5 shows the total 1 g results 28 days post one dose HbsAG.

FIG. 6 shows the total 1 g results 28 days post second dose HbsAG.

FIG. 7 shows the antibody responses (Sera ELISA) results post one dose.

FIG. 8 shows the antibody responses (Sera ELISA) post second dose.

FIG. 9a shows the antibody responses (Sera ELISA) to the and influenzaHA-Sichuan at day 14: FIG. 9b shows the antibody responses (Sera ELISA)to the anti influenza HA-Sichuan at day 28; FIG. 9c shows the antibodyresponses (Sera ELISA) to the anti influenza HA-Puerto Rico 8 (PR8) atday 14; FIG. 9d shows the antibody responses (Sera ELISA) to the antiinfluenza/a HA-Puerto Rico 8 (PR8) at day 28. The legend for die symbolsused in FIGS. 9A, 9B, 9C and 9D is shown in FIG. 9B.

FIG. 10 shows the immune response generated following immunization withformulations (Table 14) assessed by measurement of anti influenza (A/PR8strain specific) response.

DETAILED DESCRIPTION

We have formulated two related theories to explain the improvedperformance of the new compositions which are not mutually exclusive. Werefer to these theories as the general and specific theories (asdescribed below).

The General Theory

The general theory states that the enhanced performance of appropriatelyco-formulated material (wherein both DNA and its cognate protein areassociated with a single vesicle, such that vesicles in a populationhave both DNA and protein) is due to use acquisition of both nucleicacid and its cognate protein (the assistor protein) by the same antigenpresenting cell (be it a classical antigen presenting cell such as amacrophage or dendritic cell, or a B-cell, be it antigen specific ornon-antigen specific). The co-delivery of the DNA and its cognateprotein to the same cell allows a synergistic interaction in the ensuingimmune response which would not be possible if the DNA were to beacquired by one antigen presenting cell, and the protein by another.(Previously described compositions do not provide for or recognize theimportance of co-delivery or DNA and its cognate protein to the sameantigen presenting cell).

The Specific Theory

The specific theory states that the enhanced performance ofappropriately co-formulated material (wherein both DNA and its cognateprotein are associated with a single vesicle, such that vesicles in apopulation have both DNA and protein) involves the targeting (by antigenin the protein form, exposed at the surface of the particle) of thevesicle to antigen-specific B-cell thereby selectively delivering bothDNA and its cognate protean in a targeted manner to the sameantigen-specific B-cell. Since the antigen-specific B-cells willordinarily proliferate in the course of an immune response, theseproliferating cells are likely to form better targets for transductionby the nucleic acid also present in the particle than wouldnon-proliferating cells. As in the case of the general theory above, theco-delivery of the DNA and its cognate protein to the same cell allows asynergistic interaction in the ensuing immune response which would notbe possible if the DNA were to be acquired by one antigen presentingcell (in this case an antigen-specific B-cell), and the protein byanother. In the specific form of the theory, the particles are capturedby the antigen-specific receptors (i.e., surface antibodies) of B-cells,and the nucleic acid plus its cognate protein (the assistor protein) areboth taken up by individual antigen-specific B-cells.

The efficiency of nucleic-acid-based immunisation is limited by the lowtransduction efficiency that is achieved in vivo with naked formulationsof the antigen-encoding nucleic acid (e.g., naked DNA), such that fewcells take up and express the nucleic acid of interest. In theexemplification of the present invention we have used vesicular,primarily liposomal, compositions to achieve protection of the DNA fromnucleases in vivo, and to allow co-formulation of the DNA and itscognate protein (the assistor protein) in the same particle, although itshould be clear to the reader that other vesicular compositions might beused to achieve association of DNA and its cognate protein to achievethe necessary properties of co-delivery herein defined.

In a particular preferred composition according to the invention, thevesicles comprise liposomes formed from liposome forming materials,i.e., formed of lipid bilayers. The vesicles may alternatively bemono-layer. Liposomes may comprise synthetic amphiphile components, suchas surfactant type molecules. Non-ionic vesicles of this type are oftenknown as niosomes. The vesicles may not comprise phospholipids, butpreferably are based substantially on phospholipids.

In the course of our own studies using liposomal compositions weendeavoured to obtain efficient co-entrapment and/or association ofprotein and DNA in the same liposomal particles. We found that theliposomal compositions we have developed for packaging and protection ofDNA against nucleases and for DNA immunisation (as described in ourearlier case WO-A-9810748) are also very efficient at co-packagingprotein at the same time. Surprisingly, under the conditions hereindefined for formulation of the liposomes, the DNA and protein do notcompete with one another for association or containment in the liposomalparticle. Moreover, they are also capable of displaying significantquantities of the assistor protein in antigenically active form at thesurface of the liposomal particle. We believe that the surface-localisedprotein antigen of our new composition (the assistor protein) may becapable of targeting liposomes, or liposome fragments generated in vivofrom breakdown of these structures, to antigen-specific H-cells.

Although liposomally formulated DNA can be targeted to receptors onantigen presenting cells, e.g., by placing ligands for cellularreceptors of antigen presenting cells on the surface of liposomes (e.g.,mannosyl moieties or complement proteins such as C3d), antigen itselfhas not previously been used as a targeting device in nucleic acid basedvaccines.

The new compositions allow for the simultaneous presentation byantigen-presenting cells of both the acquired protein font) of theantigen (the assistor protein), plus the expressed form of the proteinfrom its cognate nucleic acid. Such composition allows a novelprime-boost effect whereby the differing kinetics of presentation of theexpressed antigenic protein and the assistor protein (having maxima atdifferent times) provide for a longer-lasting and ‘double hit’ exposureof the relevant immune cells to the antigen. Unlike other prime-boostphenomena in the nucleic arid vaccine field, the novel compositionsprovide prime and boost functions with a single dose.

Another advantageous feature of the new compositions relates to thediffering modes of antigen presentation of the two forms (the added formand the in vivo expressed forms of the protein). Since acquired andexpressed proteins are presented by two distinct pathways in antigenpresenting cells (the former resulting in peptide presentation viaclass-II MHC, the latter via class-I MHC) the new invention provides fora more broadly based immune response involving the stimulation ofT-helper cells (class-II restricted), class-II restricted effectorT-cells and cytotoxic T-cell (class-I restricted). The cellularmicroenvironment created by the new compositions (wherein both class-IIand class-I presentation are occurring at the same antigen-presentingcell surface at the same time) allows for interactions among thediffering T-cell types that engage the antigen-presenting cell. Sinceboth T-cell types (class-I and class-II restricted) can be stimulatedduring interaction with the same antigen presenting cell, and byinteractions with each other while simultaneously present it theantigen-presenting cell surface, the new formulation has severaltheoretical advantages over previously described methods andcompositions for nucleic acid immunisation. Here we describe that thetheoretical advantages are confirmed in practice at the level ofantibody responses to the antigen. We predict however that furtheradvantages will be found for the new formulation strategy in stimulatingcell-mediated immunity (including T-helper cell responses and effectorT-cell responses including cytotoxic T-cells). Since antibody responsesto protein antigens are highly T-cell dependent, the data presented inthis application on antibody production strongly suggest that the newcompositions are effective at stimulating (at lean) T-helper cell (MHCclass-II restricted) responses. The fact that the new formulationstrategy provides for simultaneous stimulation of class-II restrictedhelper and class-I restricted cytotoxic cells on the same antigenpresenting cell, also suggests that the strategy will be effective inthe stimulation of cytotoxic T-cell responses. Thus, the assistorprotein will provide additional help for cytotoxic T-cell responses,i.e., it will increase the concentration and/or duration of expressionof MHC class-II restricted peptide epitopes of the antigen recognized byT-helper cells at the surface of the antigen-presenting cell, increasingthe opportunity for T-cell help of cytotoxic T-cell responses to theexpressed form of the protein antigen presented via the class-I MHCpathway.

An immune response requires co-operation between different cells of theimmune system, namely antigen presenting cells (such as macrophages anddendritic cells, which are known as ‘classical’ antigen presentingcells). T-cells (T-lymphocytes) and B-cells (B-lymphocytes). B-cellshave a capacity to present antigens to T-cells, in a manner which is inseveral respects analogous to presentation by conventional antigenpresenting cells. It is during this B-cell T-cell interaction thatB-cells acquire the ‘help’ needed to produce specific antibodies. Unlikeclassical antigen presenting cells however, B-cells are not good atacquiring particulate antigens. Some B cells however (i.e., those whichare specific for a given antigen) are particularly good at antigenpresentation because they art able to acquire and concentrate smallantigen particles (including antigen molecules) via theirantigen-specific surface-immunoglobulin receptors. Compared tonon-antigen specific B-cells, antigen specific B-cells are al least1000× more efficient at presentation of antigen to class-II restrictedT-cell (Lanzavecchia A., Antigen-specific interaction between T and Bcells. Nature (1985) Apr 11-17:314(6011):537-539). Antigen specificB-cells may therefore be important targets for the compositions of thepresent invention, since they have the capacity to acquire particles ofantigenic protein and its associated DNA.

T-cells and B-cells recognize antigens in different ways. Thesediffering modes of recognition have implications for the differing modesof embodiment of the present invention. In order to understand thefavourable embodiments of the invention it is first necessary toappreciate the features of these differing modes of recognition. T-cellsrecognize peptide fragments of proteins embedded in class-II or class-IMHC antigens at the surface of cells, whereas B-cells (which ultimatelyproduce antibody) recognize surface features of the unfragmented antigen(which is usually protein in character), via antibody-like antigenreceptors on their cell surfaces. The differing recognition requirementsof T-cells and B-cells are reflected in the differing nature of theirepitopes. Thus whereas B-cells must recognize surface features of anantigen or a pathogen (B-cell epitopes). T-cell epitopes (which comprisepeptides of about 8-12 amino acids in length), are not obliged to bepresent on the surface of an antigen, and may be ‘internal’ as well as‘external’ when viewed in the content of the three-dimensional structureof the antigen. According to the ‘specific theory’ of the presentinvention (as defined above), the most favoured siting of a B-cellepitope is ‘surface’ on the antigen or pathogen, which facilitates theuptake and stimulation of antigen-specific B-cells by appropriatelyformulated liposomal [nucleic-acid+protein]. However, according to thegeneral theory of the Invention (where liposomes are acquired in anon-antigen-specific manner by antigen presenting cells), an epitope(particularly a T-cell epitope) may be sited internally in the structureof the antigen, and is not required lobe available or exposed on thesurface of the antigen or agent.

In one favorable embodiment of the invention both antigenic protein andassistor protein are derived from a surface antigen of a viral protein.The proteins may have the same sequences as one another or may bemutated, may have portions deleted, or may be fused with otherpolypeptides, provided that they share at least one epitope (B-cell orT-cell) preferably several.

The portions of protein which the antigenic protein and the assistorprotein have in common may be expressed in terms of their sequencehomologies. It is believed that the proteins should be such that theassistor protein has at least one contiguous sequence of at least tenresidues having at least 50%, preferably at least 75%, more preferablyat least 90%, similarity with a contiguous sequence of the same lengthof the antigenic protein. Generally the respective contiguous sequencesare at least fifty residues long, and have at least 90% sequencesimilarity, preferably at least 75% sequence similarity. More preferablythe said contiguous sequences have at least 50%, preferably at least75%, more preferably at least 90% sequence identity.

Sequence similarity is only one index of structural similarity however,and in the case of the ‘special theory’ (defined above) it is necessaryonly that the assistor protein and the encoded and expressed proteinshare a single B-cell epitope (defined as recognized by a singleantibody recognizing the agent) without any requirement for sequencehomology.

The compositions of the invention are useful for generating an immuneresponse, for instance which is sufficient to resist infection, by aninfectious agent. The antigenic protein and assistor protein are thuspreferably derived from a source which is an infectious agent, forinstance a microorganism such as a bacterium, or a virus. Suitably thevirus is a conventional virus, (as distinct from a protein-onlyinfection agent such as a prion), against which immune responses areknown (or believed to be) capable of neutralising or eliminating thevirus, for instance a hepatitis virus (such as hepatitis-B or C viruses)or an influenza virus (such as influenza-A or B viruses). Suitably theinfectious agent may be a bacterium, such us a streptococcus (e.g.,Streptococcus pneumoniae, or a member of the group-A or group-Bstreptococci) or an agent capable of causing meningitis such as themeningococci groups A, B & C or Haemophilus influenzae, or organismscapable of causing ear infections in children such as Moraxellacattharalis. Suitably the agent may be a mycobacterium such asMycobacterium tuberculosis. Suitably the antigen nay also be a hostprotein that is selectively expressed on or within cancerous cells ortissues, such as carcinoembryonic antigen, or CD55 (a complement controlprotein), or an integrin or other marker of the tumour vasculature. Thetarget antigen of this novel vaccine composition may also be a hostprotein or peptide requiring neutralisation or elimination such as aharmful autoantibody, or a peptide such as the amyloid beta peptide ofAlzheimer's disease in its various forms (A-beta 40 and A-beta 42).

The target antigen of the vaccine may also be a carbohydrate, such as abacterial polysaccharide, wherein the expressed form of the proteinand/or the assistor protein mimic(s) the antigenic structure of saidcarbohydrate antigen. Suitable peptide mimics of the group-Bmeningococcal polysaccharide (Laing, Granoff Granoff D M and Moe G R.Molecular mimetics of meningococcal B epitopes. (U.S. Pat. No.6,030,619) and of group-B streptococcal polysaccharide have beendescribed. Such peptides may be expressed as concatenated forms wherethe different peptide sequence embodiments of the carbohydrate epitopeate joined together at the DNA level to form a polypeptide or proteincomprising repeating epitopes of different sequence,

According to the present invention, the target epitope(s) against whicha beneficial immune response is desired should correspond to epitope(s)which are shared between the protein encoded by the DNA and the assistorprotein. The location of the target epitope(s) in situ on the targetantigen may be accessible to antibodies (such as neutralising epitopesof the influenza-A hemagglutinin), but alternatively may usefully alsobe internal in the agent (such as a heat shock protein of Mycobactieriumtuberculosis).

The assistor protein, though usually being highly similar or identicalto the nucleic-acid encoded protein in the composition, may be wholevirus (virion) or a ‘split’ virus preparation (such as well knowndetergent-lysed influenza virus preparations) provided that it containsa protein which has at least one (and preferably several) sharedepitopes with the nucleic-acid encoded protein. The assistor proteinwould generally not be comprised of a viable virus capable ofreplication in a host animal.

In the invention the nucleic acid may be RNA, but is preferably DNA. DNAoperatively encoding an antigen should preferably comprise a promoterand, preferably, control sequences. Suitably the DNA is plasmid DNA,conveniently derived from E coli CI plasmid, but could be linear DNA.

It may be desirable in the invention to include a nucleic acid thatencodes for more than one protein and/or to include one or moredifferent assistor proteins. For this embodiment, it is preferable thata single vesicle should have associated with it nucleic acid encodingfor an antigenic protein which shares epitopes ah described above withthe antigenic or assistor protein associated with that particle. Forexample the composition of the invention may comprise two or moredifferent liposome types in admixture, for instance one of whichcomprises nucleic acid encoding a first antigenic protein with a firstassistor protein, the antigenic protein and assistor protein beingrelated as described above, and a second liposome type comprisingnucleic acid encoding a second antigenic protein associated with asecond assistor protein related to the second antigenic protein asdescribed above. Alternatively, a single liposome may contain two ormore different nucleic acids, one of which encodes a first antigenicprotein and the other of which encode a second antigenic protein (etc.),and first and second (etc.) assistor protein, the first and secondassistor proteins being related to respective first and second antigenicproteins as described above. The two or more antigenic proteins may bepart of the same expressed protein molecule (i.e., a fusion protein),encoded by a single nucleic acid. Alternatively, two or more separatenucleic acid components, each encoding different parts of one antigenicprotein, may be included (with the protein) in a single liposome. Afavourable composition may also comprise severally co-formulated DNAsand their cognate proteins (assistor proteins), provided that each DNAis associated with its cognate or assistor protein in the same liposome.

The same arrangement of nucleic acid and protein may be achieved invesicular compositions of the invention farmed from non-phospholipidcomponents.

Embodiments involving more than one antigenic protein, as described inthe preceding paragraph, may be of particular value where thecomposition is to be used to generate an immune response, generallyvaccinate a subject, against an infective agent which may exist inseveral infective strains. This embodiment of the invention is ofparticular value where the infectious agent is a virus, especially aninfluenza virus. Thus the nucleic acid encoded antigenic proteins andtheir corresponding assistor proteins may be derived from A and Bstrains of influenza virus. One preferred embodiment would comprise twocurrently circulating (or anticipated) strains of influenza A, plus onecurrently circulating strain (or anticipated strain) of influenza-B. Thecomposition would favourably comprise all six molecular entitiesassociated with a single vesicle (e.g., a liposomal particle) such thateach particle is associated with all three nucleic acids and all threeproteins. Another favorable embodiment incorporating influenza viruseswould comprise three separately created vesicular formulationscomprising {Ai protein+AiDNA}; {Aii protein+AiiDNA} and {B protein+BDNA} (where curly brackets denote the pay-load of an individual vesicle)mixed together in a single dose or administered in three separate dosesto a recipient human or animal.

In one useful embodiment of the invention, the nucleic acid is at leastpartially, and preferably substantially wholly, entrapped within theintravesicular space of vesicles, usually liposomes. When it isentrapped in the intravesicular space, the nucleic acid is optimallyprotected from its environment, but may nevertheless be delivered intothe appropriate cells once administered to a subject. Alternatively, butless preferably, the nucleic acid may be complexed with the vesicles(hat is primarily be associated on the external surface of the vesicles.Such an arrangement provides a lower degree of protection duringadministration and delivery of the nucleic acid, but may also beeffective.

In one embodiment of the present invention the assistor protein is,preferably, at least in part, accessible at the outer surface of thevesicle. This will allow acquisition of the vesicle by antigen specificB-cells, and, following production of antibodies in the early stages ofthe immune response to the vaccine composition, will facilitate theuptake of antibody-complexed vesicles by antigen presenting cells viahigh affinity Fc-gamma receptors that recognize surface boundantigen-specific IgG on the vesicle surface, likewise, surface locatedantigen on a liposomal or other vesicle will allow complement fixation,resulting in the uptake of vesicles and their fragments by complementreceptors on antigen-presenting cells and B-cells. In order to achievethose results, the protein may be merely complexed with the externalsurface of the vesicle (e.g., by electrostatic or hydrophobicinteractions, in the manner of an extrinsic membrane protein) or,preferably, is embedded in the wall of the vesicle (e.g., via atrans-bilayer hydrophobic sequence of polypeptide chain) remainingpartly exposed to the extra-vesicle environment. In either instance,according to this embodiment, the epitope of interest should beaccessible from the outside of the vesicle. Such accessibility may bedetermined by carrying out binding experiments using antibodies againstthe respective epitope. Such binding data demonstrating surface exposureare described in the figures associated with this text for our work onco-formulation of DNA and protein for in linen/a-A virus.

Where the vesicle is liposomal, the liposome forming components used toform the liposomes may include neutral, zwitterionic, anionic and/orcationic lipid moieties. These may be used in relative amounts such asto confer an overall charge on the liposome or, less preferably, theliposomes may have no overall charge. It is found that using lipidcomponents such that the liposome has an overall positive charge canprovide good results (refer to the data section of this application). Inaddition to components which are properly termed lipids (includingglycerides and cholesterol), the liposome forming components may includenon-lipids components (i.e., which are not naturally occurring lipids)such as non-ionic or cationic surface active agents.

According to a particularly preferred embodiment of the invention, thenew composition comprises liposomes formed from liposome formingcomponents including at least one cationically charged component in anamount such that the liposomes have an overall positive charge.

In this embodiment of the invention the cationic component incorporatedinto the liposome may be any of those which have been used in liposomepreparations for improving transfection rate by complexation withpolynucleotide. The component may be a lipidic or a non lipidic compoundand may be synthetic or natural Preferred cationic lipids are1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP),1,2-bis(hexadecyloxy)-3-trimethylaminopropane (DOTMA).N-[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammoniumchloride (DOTMA) andother lipids of structure I defined in U.S. Pat. No. 4,897,355,incorporated herein by reference or the ester analogues.

The structure is as follows:

or an optical isomer thereof, wherein Y1 and Y2 are the same ordifferent and are each —O— or O—C(O)— wherein the carbonyl carbon isjoined to R1 of R2 as the case may be; R1 and R2 are independently analkyl, alkenyl, or alkynyl group of 6 to 24 carbon atoms, R3, R4 and R5are independently hydrogen, alkyl of 1 to 8 carbon atoms, aryl oraralkyl of 6 to 11 carbon atoms; alternatively two or three of R3, R4and R5 are combined with the positively charged nitrogen atom to form acyclic structure having from 5 to 8 atoms, where, in addition to thepositively charged nitrogen atom, the atoms in the structure are carbonatoms and can include one oxygen, nitrogen or sulfur atom; n is 1 to 8;and X is an ion.

Preferred embodiments are compositions wherein R1 and R2 individuallyhave from 0 to 6 sites of unsaturation, and have the structure

CH₃—(CH₂)_(a)—(CH═CH—CH₂)_(b)—(CH₂)_(c)—

wherein the sum of a and c is from 1 to 23; and b is 0 to 6. Mostpreferably each of R1 and R2 is oleyl. Particularly preferredembodiments are compositions wherein the long chain akyl groups arefatty acids, that is, wherein Y1 and Y2 are alike and are —O—C(O)—

Alternatively cationic lipids of the general structure I or the generalstructure II defined in U.S. Pat. No. 5,459,127, incorporated herein byreference may be used

Other suitable cationic compounds are the non-lipid componentstearylamine and 3β[N′N′-dimethylamino ethane)-carbarnyl]cholesterol(DC-Chol) (a lipidic component) or analogues thereof.

The liposomes, in addition to comprising cationic components, generallyalso comprise non-ionic and/or zwitterionic components which includelipids, which may be phospholipids or other lipids not includingphosphoryl groups. Preferably the lipids include phospholipids, such atnatural or synthetic phosphatidylcholines, phosphatidyl ethanolamines,phosphatidylserines in any of which the long chain alkyl groups (whichmay be joined through ester or ether linkages) may be saturated orunsaturated. Preferably the lipids groups of glyceride lipids amunsaturated. The components may include non-lipidic components, forinstance non-ionic surfactants such as sorbitan mono esters of fattyacids, and/or ethoxylated fatty acids or other analogues, such asethoxylated lanolins,

Best results are achieved when the liposomes include fusogenic lipids,which are usually phosphatidyl ethanolamines in which the acyl groupsare unsaturated. Cholesterol may be included although it may render theliposomes too stable for adequate delivery of polynucleotide into targetcells.

The amount of canonic component is preferably in the range 5 to 50% ofthe loud moles of liposome forming components, preferably in the range10 to 25% mole.

A vesicle composition is generally in the form of an aqueous suspensionfor instance, in a physiological buffer. Alternatively it could be adried composition for rehydration.

The composition is preferably a vaccine, for instance adapted foradministration by subcutaneous, intravenous, intramuscular, intradermal,nasal, oral, other mucosal or pulmonary routes.

The vesicles may be made by any of the generally used vesicle-formingtechniques. The vesicles may be multilamellar or unilamellar vesiclesand may be relatively large (vesicle diameters in the range 300 nm to5000 nm; preferably less than 2000 nm preferably with average diametersin the range 500-1000 nm), or small (vesicle diameters in the range 100nm to 400 nm preferably with avenge diameters in the range 200 to 300nm) Preferably the vesicles have a mean diameter not exceeding 500 nm,and preferably substantially all have diameters less than 2000 nm.

Although a liposomal composition of the invention may be formed byconventional liposome forming processes, such as by dispersing theliposome forming materials from a film into a suspending mediumcontaining the nucleic acid and the protein, followed by one or mote sueadjusting steps, or alternatively by co-dissolving liposome formingmaterials and nucleic acid and/or assistor protein in a common solventfollowed by a liposome forming step involving addition of aqueousliquid, or by loading nucleic acid and/or assistor protein through thewalls of preformed liposomes using concentration gradientelectroporation or electrophoretic techniques, a preferred method uses adehydration-rehydration technique.

Several suitable methods of liposomal formulation ore described in thebook-chapter by Christopher J. Kirby and Gregory Gregoriadis: ISBN0-471-14828-8 Encyclopedia of Controlled Drug Delivery Editor EdithMathiowitz, Published July 1999 by Wiley Chapter ‘L’ for liposomes.These include (non-exhaustively) multi-lamellar liposomes prepared bythe ‘hand shaken’ method; dehydration/rehydration vesicles (the methodused in the present examples, which is highly efficient); and simplehydration of solvent-solubilized lipids. The simultaneous presence ofDNA and protein in these procedures will result in various degrees ofco-entrapment and other forms of association of both entities with theliposomes. Calcium phosphate may also be used to precipitate DNA andprotein together resulting in a ‘protein co-formulation with DNA’version of our invention described in WO-A-0141739.

Another favored method for the formation of associated DNA and itscognate protein is that published by Judith Senior and GregoryGregoriadis (Biochimica et Biophysica Acta (1989) 1003;58-62). This is avariant of the dehydration-rehydration method wherein the ‘assistorprotein’ component of the present invention may be incorporated bycovalent conjugation onto the surface of small unilamellar vesicles(SUV). Such SUV are then lyophilised, and then re-hydrated according toSenior and Gregoriadis (above), in a solution of the antigen-encodingDNA. The resulting multi-lamellar vesicles have most of the proteinpayload on the surface of the liposomal particle, which is a favouredembodiment of the present invention.

A process according to the invention for forming a liposomal compositioncomprises the steps

-   -   a) providing an aqueous suspension of small unilamellar vesicles        (SUVs) formed of liposome forming materials;    -   b) contacting the aqueous suspension of SUVs with nucleic acid        which operatively encodes tin antigenic protein to form an        SUV-nucleic acid suspension;    -   c) dehydrating the SUV-nucleic acid suspension to provide a        dehydrated mixture; and    -   d) rehydrating the dehydrated mixture in an aqueous resuspending        medium to form a suspension of nucleic acid containing liposomes    -   including the step of introducing an assistor protein whereby        the nucleic acid containing liposomes are associated with said        assistor protein.

The dehydration-rehydration method results in nucleic acid beingentrapped within the intra vesicular space of the product liposomes.Additionally a small amount may be left on the outside of the liposomes.The assistor protein may be added at various different stages of theprocess. It may be contacted with the aqueous suspension of SUVs before,during or after step b and before step c. The assistor protein willbecome coentrapped within the intravesicular space of the liposomes withnucleic acid.

In an alternative process, the assistor protein is present in theresuspending medium during the rehydration step. In this embodiment, atleast a pan of the protein is likely to be exposed on the externalsurface of the liposomes. In an alternative process, the protein may becontacted with the aqueous suspension of nucleic acid containingliposomes. This embodiment will result in substantially all of theprotein being associated with the external surface of the liposomes.

In order to increase the degree of incorporation of protein, whilststill allowing exposure of epitopes at the external liposome surface, itmay be desirable in some embodiments to conjugate the assistor proteinto a lipophilic moiety which is suitable for embedding within the wallof the liposome, such as a fatty acyl moiety. The conjugation maycomprise a part of the preparation procedure for the assistor protein.Alternatively, the assistor protein may be chemically conjugated to acomponent of the liposome after step d.

Where the liposome forming materials include cationic moiety such thatthere is an overall cationic charge on the liposomes, there may beadequate electrostatic attraction between the positively chargedliposomes and the assistor protein, where this has an overall negativecharge under the ambient conditions such that hydrophobic protein isneeded and complexation of the protein provides a strong enoughassociation.

Preferably the liposome funning materials comprise at least 5% by molecationic compound.

In the invention the weight ratio of nucleic acid to assistor protein ispreferably in the range 1000:1 to 1:1 most preferably the ratio is in arange between ratio 5:1 and 3:1.

The weight ratio of nucleic acid to liposome forming material preferablyin the range 1:100 to 1:1000 more preferably in the range of 1:100 to1:300.

In the process of the invention, the liposomal particles used in step a)preferably have sizes in the range 30 nm to 5000 nm, most preferablysubstantially all of the liposomes having diameters less than 1000 nmThe process results in product liposomes having particle sizes in therange 200 nm to 5000 nm, preferably in the range 300 nm to 2000 nm.Where necessary, the process may involve a size-controlling feature.This may involve incorporation of components into the re-suspendingmedium which control the liposome size (such as sugars, as described inWO-A-0156548). Alternatively the fee control may involve an additionalstep following step d, in which the suspension is subjected tomicrofluidisation, passage through filters or homogenisation. Sonicationis a loss preferred but viable option for this purpose but it resultsinevitably in some level of DNA fragmentation.

After the process, it is preferable for the product liposomes,comprising both nucleic acid and protein, to be subjected to apurification step, in which non-entrapped nucleic, or assistor protein,or other components, are removed from the product suspension. Suchpurification processes may involve centrifugal ions, filtration, passagethrough a porous membrane of defined pore size, gel-exclusionchromatography, such as sue exclusion chromatography, where the vesiclesappear In the void volume.

We have found that the present invention is highly effective forgenerating an immune response when administered to a subject,particularly an improved antibody response. We believe the improvementexhibited by the present invention to be due fundamentally to theco-targeting of nucleic acid and assistor protein to the same antigenpresenting cells, (possibly including antigen specific B-cells), suchthat following encounter with a suitably formulated vesicle anindividual antigen presenting cell takes up both the nucleic acid andits cognate protein. In the case of the influenza hemagglutinin, weobserve that separate formulation of nucleic acid (DNA encodinghemagglutinin) and its cognate protein (hemagglutinin protein) inseparate liposomal compartments or populations, followed by mixing andco-administration in vivo, does not achieve the synergistic effect ofco-formulation of the DNA and its cognate protein in the same liposomalparticles such that each liposome contains both DNA and its cognateprotein. These data support our hypothesis that the synergy of DNA withits cognate protein in eliciting an immune response (in this caseagainst the influenza hemagglutinin) requires the appropriateformulation to allow co-targeting of both DNA and its cognate protein tothe same antigen presenting cell.

EXAMPLES

The present invention is illustrated in the accompanying examples:

Example 1 Haemagglutinin in Cationic Liposomes Materials and Methods:Lipids

Egg phosphatidylcholine (PC), Dioleoyl phosphatidyl-ethanolamine (DOPE)and 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) were purchasedfrom Sigma Chemical Co., UK. All lipids were stored (−20° C.) dissolvedin chloroform, purged with nitrogen

DNA

Plasmid pCI-OVA (ref DNA OVA) (a kind gift of Dr. T. Nagata, HamamatsuUniversity School of Medicine, Japan) contains the chicken egg albuminprotein (ovalbumin, OVA) (Yoshida A. Nagata T, Uchijima, Higashi T, andKoide Y. “Advantage of gene gun-mediated over intramuscular inoculationof plasmid DNA vaccine in reproducible induction of specific immuneresponse.” Vaccine (2000) 18:1725-1729) cDNA cloned at the EcoR1 site ofthe pCI plasmid (Promega, Madison, Wis.) downstream from the CMVenhancer/promoter region. Plasmid p1.17/SichHA (ref DNA HA) was providedby Dr. J. Robertson (NIBSC, UK) (Johnson. P. et al., J. Gen. Virol.(2000) 1737-1745) containing the full length HA from influenzaA/Sichuan/2/87. Both plasmids for dosing were commercially produced byAldevron (Fargo, USA) and contained <100 Endotoxin Unit (EU)/mg of DNAwith no residual protein detectable.

Proteins

Influenza A/Sichuan/2/87 whole inactivated virus protein (sucrosegradient purified, major protein HA, ref antigen HA) was obtained fromthe NIBSC, UK, Ovalbumin (Grade VI, ref antigen OVA) were purchased fromSigma Chemical Co., UK.

Preparation of Liposome Compositions

Briefly, small unilamellar vesicles (SUV) were prepared from eggphosphatidylcholine (PC) and dioleoyl phosphatidylcholine (DOPE) and1,2-dioleoyloxy-3-(trimethylammionium) propane (DOTAP) (4:2:1 molarratio) by sonication were mixed with DNA or protein alone or DNA andprotein together (Table 1). Formulations were prepared in triplicate,two vials for dosing (prime and boost) undone vial for % entrapmentcalculations based radio labeled tracer (HA and OVA, DNA and protein)added to materials for entrapment and freeze-dried overnight asdescribed in Gregoriadis, G., Saffie, R. and Hart, S. L., “High yieldincorporation of plasmid DNA within liposome: effect on DNA integrityand transfection efficiency.” J Drug Targeting (1996) 3:467-475 and inKirby, C., Gregoriadis, G., “Dehydration-rehydration vesicles (DRV): Anew method for high yield drug entrapment in liposomes”. Biotechnology(1994) 2:979-984. Following rehydration under controlled conditions, thegenerated dehydrated-rehydrated vesicles (DRV liposomes) were washed bycentrifugation to remove non-incorporated DNA. The washed pellets wereresuspended in PBS to the required dose volume. DNA and/or proteinincorporation into liposomes was estimated on the basis of ³⁵S (for DNA)and ¹²⁵I (for protein) radioactivity recovered in the suspended pellets.Liposomes were subjected to microelectrophoresis and photon correlationspectroscopy (PCS) at 25° C. in a Malvern Zetasizer 3000 to determinetheir /eta potential (ZP) and z-average diameter respectively.

TABLE 1 Formulation % Antigen Liposome Entrapment Group DNA μg μg(PC:PE:DOTAP μmole) DNA Protein 1.1 70 (HA) 4.2 (HA) 11.2:5.6:2.8 10099.5 1.2 70 (OVA) 4.2 (HA) 11.2:5.6:2.8 91.9 84.1 1.3 70 (HA) 5.25 (OVA)11.2:5.6:2.8 97.5 99.0 1.4 70 (HA) 11.2:5.6:2.8 100 — 1.5 4.2 (HA)11.2:5.6:2.8 — 89.8

Animal Procedures

Female Balb/c mice 6-12 weeks old (Harlan, UK) were immunised bysubcutaneous injection administered in 0.2 ml dose volume (table 2).Final dose quantities were calculated based on % material (DNA, Proteinor both) entrapped (from radioactivity count vials). Negative controlmice received doses of PBS respectively. Mice received two doses ofantigen at days 0 and 28, with sample bleeds collected from the tailvein at day 16, 28 and 42 with respect to the first injection.

Formulation Capture ELISA

Formulations for immunisation (Table 2) were assayed by captureenzyme-linked immunoadsorbent assay (ELISA) for HA (A/Sichuan)antigenicity. Certified binding chemistry 96 well plates were coatedovernight at 4° C. with 50 μl/well of 1:2000 dilution of sheep antiA/Sichuan HA reference sera (NIBSC) in carbonate buffer (pH 9.6). Afterremoving the sheep antibody solution, wells were coated with 200 ul of10% (w/v) FCS (Fetal Calf Sen) in PBS. After 2 h at 37° C., the blockingsolution was removed and serially diluted (×2 series) formulations (refTable 2) were added to the wells (50 μl sample/well). Formulations werediluted in PBS and Triton X100 (Tx-100) which is capable by lysingliposomal formulations to reveal entrapped materials (5) Gregoriadis, G,Brenda McCormack, Mia Obrenovic, Roghieh Saffie, Brahim Zadi, and YvonnePerrie, “Vaccine entrapment in liposomes”, Methods (1999) 19:156-162).Following 1 h incubation at 37° C., plates were washed four times withPBS/Tween 20 and overlaid with dilutions of a murine specific (influenzaA/Sichuan) antisera at a dilution 1/5000 (50 μl sample/well). Hollowing1 h incubation at 37° C. plates were washed four times with PBS/Tween 20and overlaid 50 μl/well of rabbit anti-mouse Ig-HRP conjugated sera(Dako). After 1 h at 37° C., plates were washed four times withPBS/Tween 20 and overlaid with 50 μl/well of substrate solutiono-phenylenediamine (Sigma, Fast OPD). The reaction was stopped by adding50 μl/well of slopping solution (3M Sulphric Acid) and the adsorbance ofeach well at 490 nm was determined.

Sera ELISA

Sera obtained form sample bleeds were diluted 20-fold in PBS and kept at−20 EC. until assayed by the enzyme-linked immunoadsorbent assay(ELISA). Certified binding chemistry 96-well plates were coaledovernight at 4° C. with 50 μl/well of 1:1000 dilution of sheep antiA/Sichuan HA reference sera (NIBSC, UK) in carbonate buffer (pH 9.6).After removing the sheep antibody solution, wells were coated with 200μl of 2% (w/v) BSA in PBS. After 2 h at 37° C., the blocking solutionwas removed and Influenza A/Sichuan/2/87 whole inactivated virus protein(sucrose gradient purified, major protein HA) 2.5 ug/ml (in PBS) wereadded to the wells (50 μl sample/well). Following 1 h incubation at 37°C. plates were washed four times with PBS/Tween 20 (trade mark) andoverlaid with dilutions of the different experimental serum (individualanimal sample bleeds or group sera pools) starting at dilution 1/100 (50μl sample/well). Allowing 1 h incubation at 37° C., plates were washedfour times with PBS/Tween 20 and overlaid 50 μl/well of rabbitanti-mouse Ig-HRP conjugated sera (Dako). After 1 h at 37° C., plateswere washed four times with PBS/Tween 20 and overlaid with 50 μl/well ofsubstrate solution o-phenylenediamine (Sigma, Fast OPD). The reactionwas stopped by adding 50 μl/well of stopping solution (3M sulphuricacid) and the absorbance of each well at 490 nm was determined. Theantibody response was expressed as the reciprocal serum dilutionrequired for OD to reach a reading of 0.200 (end point dilution). Seraconversion criteria were established from negative control animals (seeTable 3, group 12 responses), ×2 negative control (OD approximately 0.2units).

TABLE 2 Dose Formulations Dose/animal (0.2 ml subcutaneous) GroupLiposome DNA (ug) Antigen (ug) 1.1  Yes (co formulated) HA (10) HA (0.6)1.2  Yes (co formulated) OVA (11) HA (0.6) 1.3  Yes (co formulated) HA(10) OVA (0.76) 1.4  Yes HA (10) Nil 1.5  Yes Nil HA (0.6) 1.6  Yes(admix 1.4 and 1.5) HA (10) HA (0.6) 1.7  Nil HA (10) OVA (0.76) 1.8 Nil OVA (11) HA (0.6) 1.9  Nil HA (10) HA (0.6) 1.10 Nil HA (10) Nil1.11 Nil Nil HA (0.6) 1.12 Nil Nil Nil

Results

These data show that the compositions give rise to highly efficientco-entrapment of DNA and protein, the presence of protein having only aminor negative effect on the efficiency of entrapment of DNA and viceversa.

Assessment of formulations for HA (Influenza A/Sichuan strain)antigenicity are shown in FIG. 1, OD490 nm signal is proportional to HAantigen. The sera antibody results for the twelve groups (Table 2) areshown in Table 3. The results are also illustrated in FIG. 2 (clay 16).FIG. 3 (day 28) and FIG. 4 (day 42), day 15 following the second dose).

TABLE 3 Group Formulation Total Ig anti A/Sichuan influenza antigen 5DNA Antigen 16 d post 1 dose 28 d post 1 dose 15 d post 2 doses mice/grp10 ug/dose 0.6 ug/dose OD (+/−sem) sero/5 Titre OD (+/−sem) sero/5 TitreOD (+/−sem) sero/5 Titre 1.1.1 Lip( HA HA) 0.400 (0.017) 5 675 0.457(0.053) 5 1298 1.181 (0.071) 5 6015 1.2.1 Lip( OVA* HA) 0.142 (0.039) 1<100 0.240 (0.017) 4 150 0.656 (0.034) 5 1847 1.3.1 Lip( HA OVA)** 0.090(0.012) 0 <100 0.073 (0.003) 0 <100 0.158 (0.014) 0 <100 1.4.1 Lip( HAnil) 0.086 (0.013) 0 <100 0.070 (0.007) 0 <100 0.126 (0.026) 0 <1001.5.1 Lip( nil HA) 0.075 (0.013) 0 <100 0.132 (0.043) 1 <100 0.425(0.055) 5 974 1.6.1 Lip(HA) +Lip(HA) 0.085 (0.009) 0 <100 0.158 (0.034)2 <100 0.468 (0.042) 5 477 1.7 nil HA +OVA 0.067 (0.004) 0 <100 0.080(0.004) 0 <100 0.176 (0.009) 1 <100 1.8 nil OVA +HA 0.130 (0.046) 1 <1000.279 (0.122) 2 191 0.517 (0.253) 4 423 1.9 nil HA +HA 0.079 (0.007) 0<100 0.099 (0.011) 0 <100 0.381 (0.104) 4 334 1.10 nil HA nil 0.046(0.012) 0 <100 0.007 (0.002) 0 <100 0.180 (0.009) 1 <100 1.11 nil nil+HA 0.078 (0.014) 0 <100 0.083 (0.006) 0 <100 0.336 (0.096) 4 298 1.12nil nil nil 0.115 (0.013) 0 <100 0.050 (0.002) 0 <100 0.063 (0.008) 0<100 OD determined at 1/100 sera dilution, sero (=sero conversion) >0.2OD units at 1/100 sera dilution, Titre = dilution sera yielding OD Value0.2 OD units (measured on pool of individual sera/group) *dose 11 μg*dose 0.76 μg

Discussion

Assessment of the formulations (Table 2) for HA antigenicity by captureELISA (FIG. 1) was preformed on formulation in the absence and presenceof Triton X100 a liposome disrupting agent (note:-groups 1.3, 1.4, 1.7,1.10 and 1.12 serve as negative controls for the assay as theseformulations do not contain HA protein). Formulations tested in theabsence of TX100 indicate HA antigen readily delectable in formulationscontaining HA in which the protein is not formulated with liposomes(Grps 1.8, 1.9, 1.10), with a small HA antigen positive signaldetectable for formulations 1.1 and 1.2, presumably generated by surfaceexposure of HA antigen capable of being bound by antibodies employed inthis assay. In the presence of TX100 a substantially greater positivesignal is obtained for liposomal groups 1.1 and 1.2, indicatingdetection of antigen previously (cf without TX100) contained within theLiposomal formulation. Whilst formulation 1.5 and 1.6 both contain HAantigen entrapped by Liposomes in the absence of DNA in the formulation(cf 1.1 and 1.2) little HA antigenicity can be resolved.

The immune response generated following immunization with formulations(Table 2) was assessed by measurement of anti influenza (major proteinHA) antibody response. Results are summarized in Table 3 and alsoillustrated in FIGS. 2, 3 and 4. Formulation 1.1 which consisted of bothHA DNA and protein co-delivered in the same liposomal formulationproduces a greater response than all the other formulations at each serasample bleed tested (day 16, day 28 and day 42 (day 15 following thesecond dose)). The response for this co-delivered formulation is greaterin terms of magnitude (OD490 nm 1/100 dilution sera and Titre) andnumber of animals deemed sero positive at each bleed point.

Formulations 1.2 and 1.3 which also consist of co delivered protein andDNA in the same liposomal formulation fail to generate as substantialresponse as formulation 1.1. formulations 1.2 and 1.3 consist of proteinand DNA non cognate to each other.

Formulation 1.6 provides-further indication of this invention, in thatin terms of protein and DNA product administered to the animalformulation 1.1 is equivalent to 1.6. However in formulation 1.1 theproducts are co delivered in the same liposomal vehicle whilst informulation 1.6 the DNA and protein are delivered in separate liposomalvehicles. Again as stated previously the immune response to formulation1.1 is substantially greater than 1.6.

The response to HA DNA containing formulations, excluding 1.1,(Formulations 1.3. 1.4, 1.7 and 1.10) essentially fail to genomic animmune response, however this response may be dose related as Johnson.P., et al., J. Gen. Virol. (2000) 1737-1745 have reported positiveresponse to this plasmid following immunisation (non-entrapped).

The adjuvant effect of liposomes for protein delivered in liposomalformulations (Formulation 1.5 versus Formulation 1.11) previouslyrepented (Gregoriardis G, Tan L, Ben-Ahmeida ET, Jennings K., Vaccine(1992) 10(11):747-753) is weakly visible in the 15d post 2 dose samplebleed and again dose and immunisation schedule differences may accountfor different experimental results. The immunoadjuvant action of plasmidDNA in liposomes previously reported (Gursel M. et al., Vaccine (1999)17:1376-1383) is also demonstrated in the difference in responses toformulation 1.2 and 1.5. However, the synergistic effect ofco-formulation of hemagglutinin protein with its appropriate (cognate)plasmid far exceed any effect attributable to the well-knownimmunoadjuvant effects of DNA such as those observed by Klinman(Klinman. D., et al., Vaccine (1999) 19:25 19-26) for CpG motifs.

Whilst the results presented are obtained following subcutaneousadministration of the liposomal formulation, the proposed mechanisms ofaction, general and specific (plus antigen kinetics properties), shouldbe effective by alternative routes of administration; intravenous,intra-muscular, intradermal, via nasal/pulmonary and oral routes, etc.Indeed liposomes have been successfully used by these routes to deliverboth proteins and DNA, and generate immune response.

In summary we have found that the present invention is highly effectivefor generating an immune response when administered to a subject. Theresponse involves an antibody response. The improvement exhibited by thepresent invention involves composition of liposome forming materialsand, associated with the liposomes, nucleic acid operatively encoding anantigenic protein and a co-delivered protein, wherein the co deliveredprotein shares epitopes with the antigenic protein.

Example 2 Mannose Targeted Hepatitis B Vaccine Materials and MethodsLipids

Egg phosphatidylcholine (PC), Dioleoyl phosphatidyl-ethanolamine (DOPE)and 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) were purchasedfrom Sigma Chemical Co., UK. N-[2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-ylsulfanyl)-propionamide(formula given below (DOGP4αMan, a kind gift from Dr. Francis Schuber(Universitë Louis Pasteur Strasbourg I, Faculté de Pharmacie 74, routedo Rhin, BP 24 67401 Illkirch Graffenstaden)). All lipids were stored(−20° C.) dissolved in chloroform, purged with nitrogen.

Plasmid pRc/CMV-HBs(S) (or simply pCMV-S) expresses the hepatitis Bsurface antigen (small, or S, protein) under the control of the CMVimmediate-early promoter (Davis, H. L., Michel. M. L., Whalen, R. G.,“DNA-based immunization for Hepatitis B induces continuous secretion ofantigen and high levels of circulating antibody.” Human MolecularGenetics (1993) 2:1847-1851). Plasmid for dosing was commerciallyproduced by Aldevron (Fargo, USA) and contained <100 Endotoxin Unit(EU)/mg of DNA with no residual protein detectable

Proteins

Hepatitis U Surface Antigen (HBsAg) Recombinant protein, Purity: >95% bySDS-PAGE, purified from yeast Hansenula polymorpha (purchased fromAldevron, Fargo, USA. Lot 05/00 HBsAg).

Preparation of Liposome Composition

Briefly, small unilamellar vesicles (SUV) were prepared from eggphosphatidylcholine (PC) and dioleoyl phosphatidylcholine (DOPE),1,2-dioleoyloxy-3-trimethyl-ammonium) propane (DOTAP) and (DOTAP) andN-[2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}-ethoxy)ethyl]-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-ylsulfanyl)-propionamide)(DOGP4αMan) (4;2;1:1 molar ratio) by sanitation were mixed wish DNA andprotein alone or DNA and protein together (Table 4). Formulations wereprepared in two vials for dosing (prime and boost) each vial containedradio labeled tracer (DNA and protein materials) added to materials tobe entrapped (for % entrapment calculations) and freeze-dried overnightas described in Gregoriadis, G., Saffie, R. and Hart, S. L., High yieldincorporation of plasmid DNA within liposome: effect on DNA integrityand transfection efficiency, J Drug Targeting. 1996, 3(6), 467-475 andin Kirby, C., Gregoriadis, G. Dehydration-rehydration vesicles (DRV): Anew method for high yield drug entrapment in liposomes. Biotechnology,1994, 2,979-984. Prior to freezing (pre freeze dry process) sucrose wasadded to each vial at an lipid to sucrose ratio (w/w) of 1:3 and allowedto dissolve at RT (Brahim, Z. and Gregoriadis, G. A novel method forhigh-yield entrapment, of solutes into small liposomes, J LiposomeResearch. 2000, 100(1), 73-80). Following rehydration under controlledconditions, the generated dehydrated-rehydrated vesicles (DRV liposomes)were diluted in PBS to the required dose volume. A volume (˜25%) of eachvial following rehydration was washed by centrifugation to removenon-incorporated materials. The percentage incorporate of DNA and/orprotein into the liposomal formulation was estimated on the basis of ³⁵S(for DNA) and ¹²⁵I (for protein) radioactivity recovered in thesuspended pellets. Liposomes were subjected to microelectrophoresis andphoton correlation spectroscopy (PCS) at 25° C. in a Malvern Zetasizer3000 to determine their zeta potential (ZP) and z-average diameterrespectively.

TABLE 4 Formulation DNA Protein Liposome Group μg (HbsAg) μgPC:DOPE:DOTAP:DOG4αMan μM 2.1 464 13.28 42.6:21.3:5.3:5.3 2.2 nil 13.2842.6:21.3:5.3:5.3 2.3 464 Nil 42.6:21.3:5.3:5.3

Animal Procedures

Female Balb/c mice 6-12 weeks old (Harlan, UK) were immunised bysubcutaneous injection administered in 0.2 ml dose volume. Final dotequantities are summarised in Table 5. Mice received two doses of antigenat days 0 and 28, with sample bleeds collected from the tail vein at day(post 1 dose) and (post 2 doses) with respect to the first injection.

TABLE 5 Dose Quantity Total/animal Group DNA μg Protein (HBsAg) μg Lipidmg 2.1 35 1 4.35 2.2 nil 1 4.35 2.3 35 nil 4.35

Sera ELISA

Sera obtained form sample bleeds were diluted in PBS and kept at −20° C.until assayed by the enzyme-linked immunoadsorbent assay (ELISA).Certified binding chemistry 96-well plates were coated overnight at 4°C. with 50 μl/well of Hepatitis B Surface Antigen (HBsAg) Recombinantprotein at 2.5 μg/ml (Aldevron, Fargo, USA. Lot 05/00 HBsAg) in 0.1Mcarbonate buffer (pH 9.6). After overnight incubation wells were washedfour times with PBS/Tween 20™ (PBST) then wells were coated with 200 μlof 2% (w/v) BSA in PBS. After 2 h at 37° C., the blocking solution wasremoved and wells were washed four times with PBST and overlaid withdilutions of the different experimental serum (individual animal samplebleeds) starting at dilution 1/100 (50 μl sample/well). Following 1 hincubation at 37° C., plates were washed four times with PBST andoverlaid 50 μl/well of rabbit anti-mouse Ig-HRP conjugated sera (Dako).After 1 h at 37° C., plates were washed four times with PBST andoverlaid with 50 μl/well of substrate solution3,3′,5,5′tetramethyl-benzidine (TMB, Pierce). The reaction was stoppedby adding 50 μl/well of skipping solution (2M sulphuric acid) and theabsorbance of each welt at 450 nm was determined.

The physical characteristics (% product (DNA and/or protein) entrapment,particle size and surface potential (Zeta)) are summarized in Table 6.

TABLE 6 Dose 1 Dose 2 Dose 1 Dose 2 % entrapment % entrapment Size ZetaSize Zeta Group DNA Protein DNA Protein nm mV nm mV 2.1 79.8 71.3 90.588.9 379 18.1 448 ND 2.2 nil 30.3 nil 58.3 166 18.5 134 ND 2.3 85.6 nil94.5 nil 367 19.1 355 ND ND = not determined.

The antibody responses (Sera ELISA) are expressed as the mean (n=5animals/group) OD signal±SEM at the Log₁₀ serum dilution assayed. Resultare expressed in FIG. 5, which shows the total 1 g results 28 days postone dose HbsAG, and FIG. 6, which shows the total 1 g results 28 dayspost second dose.

Discussion

The use of mannose ligand targeted liposomes as delivery vehicles forDNA and protein for induction of an immune response has been describedpreviously described (Kawakami S, Sato A, Nishikawa M, Yamashita F,Hashida M, Gene Ther. (2000) 7(4):292-299, and Latif N, Bachhawat B K,Immunol Lett (1984) 8(2):75-78). Moreover the general utility of mannosereceptor mediated uptake of antigen(s) (proteins) by antigen presentingcells is recognised as a powerful component in the induction of animmune response (Lanzavecchia, A., Curr Opin Immunol (1996) 8:348-354).The use of mannose ligand targeted liposomes to co-deliver both DNA andprotein, within the same delivery vehicle (and likely to the same targetcell) has not been reported.

The immune response generated following immunization with formulations(Table 4 and 5) was assessed by measurement of anti Hepatitis B SurfaceAntigen (HBsAg) antibody response. Results are illustrated in FIGS. 5and 6). Formulation 2.1 which consisted of both HA DNA and proteinco-delivered in the same liposomal formulation produces a greaterresponse than all the other formulations at each sera sample bleedtested (day 28 and day 56 (day 28 following the second dose)). Theresponse for this co-delivered formulation is greater in terms ofmagnitude of both OD450 nm sera dilution and titre (endpoint read OD 0.3units).

The response to the DNA containing formulations, excluding 2.1,Formulation 2.3, generate an immune response consistent with publishedresults (Gregoriadis G. Pharm Res. (1998) 15(5):661-670)using the sameDNA product (at 10 μg DNA dose) in the same liposomal vehicle(PC:DOPE:DOTAP), without the mannose lipid (DOGP4αMan) component. Theimmunoadjuvant action of plasmid DNA in liposomes has been previouslyreported (Gursel, M., et al., Vaccine (1999) 17:1376-1383) using thesame DNA (non encoding, ISS capacity control) and protein products asdescribed herein. The reported adjuvant action of the DNA component forantigen-pDNA co-entrapped formulation in this paper is described asmodest, at approximately 3 fold (Titre, post 2 doses results). Thesimilar result exemplified (in FIG. 6), when a encoding (HBsAg) DNAcomponent is used (Formulation 2.1) shows a 30 fold increase in response(cf Formulation 2.2). Thus the synergistic effect of co-formulation ofHBsAg protein with its appropriate (cognate) plasmid exceeds any effectattributable to the immunoadjuvant effects of DNA, approximately only 3fold, such as those observed by Gursel or Klinman (Klinman, D, et al.,Vaccine (1999) 19:25 19-26) for CpG motifs alone.

In summary we have found that the present invention is highly effectivefor generating an immune response when administered to a subject. Theresponse involves an antibody response. The improvement exhibited by thepresent invention involves composition of liposome forming materialsincluding a mannosylated lipid component and, associated with theliposomes, nucleic acid operatively encoding an antigenic protein and aco-delivered protein, wherein the co-delivered protein shares epitopeswith the antigenic protein.

Example 3 Protection from Influenza Virus Challenge Material and MethodsLipids

Egg phosphatidylcholine (PC), Dioleoyl phosphatidyl-ethanolamine (DOPE)and 1,2-dioleoyl-3-trimethylammonium) propane (DOTAP) were purchasedfrom Sigma Chemical Co., UK. All lipids were stored (−20° C.) dissolvedin chloroform, purged with nitrogen.

DNA

pL18/PR8-HA (ref DNA HA) was provided by Dr. J. Robertson (NIBSC, UK)containing the full length HA from influenza A/Puerto Rico/8/34. Plasmidfor dosing was commercially produced by Aldevron (Fargo., USA) andcontained <100 Endotoxin Unit (EU)/mg of DNA will no residual proteindetectable.

Influenza A/Puerto Rico/8/34 whole inactivated virus protein (sucrosegradient purified, major protein HA, ref antigen HA) was obtained fromthe NIBSC, UK.

Preparation of Liposome Composition

Briefly, small unilamellar vesicles (SUV) were prepared from eggphosphatidylcholine (PC) and dioleoyl phosphatidyl-choline (DOPE) and1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP) (4:2:1 molarratio) by sonication were mixed with DNA (ref DNA HA) and protein (refantigen HA) see Table 7. Formulations were prepared in quadruplicate,two vials for dosing (prime and boost) and two vial for % entrapmentcalculations based radio labeled tracer (HA; DNA and protein) added toentrapped materials and freeze-dried overnight as described inGregoriadis, G., Saffie, R. and Hart, S. L., “High yield incorporationof plasmid DNA within liposome: effect on DNA integrity and transfectionefficiency.” J Drug Targeting (1996) 3(6):467-475 and in Kirby, C.,Gregoriadis, G., “Dehydration-rehydration vesicles (DRV): A new methodfor high yield drug entrapment in liposomes.” Biotechnology (1994)2:979-984. Following rehydration under controlled conditions, thegenerated dehydrated-rehydrated vesicles (DRV liposomes) were washed bycentrifugation to remove non-incorporated DNA. The washed pellets wereresuspended in PBS to the required dose volume. DNA and/or proteinincorporation into was estimated on the basis of ³⁵S (for DNA) and ¹²⁵I(for protein) radioactivity recovered in the suspended pellets.Liposomes were subjected to microeletrophoresis and photon correlationspectroscopy (PCS) at 25° C. in a Malvern Zetasizer 3000 to determinetheir zeta potential (ZP) and z-average diameter respectively.

Animal Procedures

Female Balb/c mice 6-12 mice old (Harlan, UK) were immunised bysubcutaneous injection administered in 0.2 ml dose volume. Final dosequantities are summarised in Table 7. Mice received two doses on days 0and 28, with sample bleeds collected from the tail vein at day 21 (post1 dose) and 42 (post 2 doses) with respect to the first injection.

TABLE 7 Group Dose Quantity Total/animal (formulation) DNA μg Protein(HA) μg Lipid mg 3.1 10 1.5 2.1 3.2 10 0.5 2.1 3.3 10 1.5Non-liposomally delivered, admixed (DNA + Protein) 3.4 nil nil nil (PBS)

Live Influenza Virus Challenge

Mice were challenged at day 57, with respect to the first immunization,with approximately 10 MID₅₀ (50% mouse infective doses) in PBS with 2%(w/v) BSA of an mouse adapted live influenza virus (A/Puerto Rico/8/34)at the National Institute of Biological Standards and Controls, UK(NIBSC). The virus was administered to non-anaesthetized mice in 50 μlvolumes bilaterally by intranasal instillation. At daily intervals alterchallenge, nasal washes were performed using 0.5 ml PBS with 2% (w/v)BSA per mouse. The presence of shed influenza virus is nasal washsamples was assessed immediately after sampling. Nasal wash samples inserum-free Eagle's minimal essential medium were plated on TPCK-trypsintreated confluent monolayers of MDCK cells is 96-well tissue cultureplates. After incubation for 3 days at 35° C., the presence of virus ineach well was determined by incubation of 50 μl supernatant with anequal volume of 0±7% (v/v) turkey red blood cells. Virus positive sampleproduced visible haemagglutination (agglutination spot clearly visible).

Sera ELISA

Sera obtained form sample bleeds were diluted in PBS and kept at −20° C.until assayed by the enzyme-linked immunosorbent assay (ELISA).Certified binding chemistry 96-well plates were coated overnight with 50μl/well of Influenza HA-PR8 antigen (20 μg/ml) in PBS. Incubateovernight at 4° C. After overnight incubation wells were washed fourtimes with PBS/Tween 20™ (PBST) then wells were coaled with 200 μl of 2%(w/v) BSA in PBS. After 2 h it 37° C., the blocking solution was removedand wells were washed four times with PBST and overlaid with dilutionsof the different experimental serum (individual animal sample bleeds)surfing at dilution 1/100 (50 μl sample/well). Following 1 h incubationat 37° C., plates were washed four times with PBST and overlaid 50μl/well of rabbit anti-mouse Ig-HRP conjugated sera (Dako). After 1 h at37° C. plates were washed four times with PBST and overlaid with 50μl/well of substrate solution 3,3′,5,5′tetra-methylbenzidine (TMB,Pierce). The reaction was stopped by adding 50 μl/well of sloppingsolution (2M sulphuric acid) and the absorbance of each well at 450 nmwas determined.

Results

The liposomal physical characteristics (% product DNA and/or protein)entrapment, particle size and surface potential (Zeta)) are summarizedin Table 8.

TABLE 8 Dose 1 Dose 2 Dose 1 Dose 2 % entrapment % entrapment Size ZetaSize Zeta Group DNA Protein DNA Protein nm mV nm mV 3.1 80.0 78.9 89.391.1 913 ND 820 43 3.2 85.6 81.5 92.2 91.0 821 ND 906 43 ND = notdetermined.

The antibody responses (Sera ELISA) are expresses;! as the mean (n=5animals/group) OD signal±SEM at the Log₁₀ serum dilution assayed.Results are expressed in FIG. 7, which shows the response after one doseand FIG. 8 which shows the results after two doses.

The live virus challenge results are presented in Table 9, as thepercentage of animals n=15 challenged)/group which presented detectablevirus in nasal wash samples obtained.

TABLE 9 Group Day 1 Day 2 Day 3 Day 4 Day 5 3.1 0 0 3.3 30 3.6 3.2 6.76.7 53.3 50 3.3 3.3 0 35.7 39.3 60.7 50 3.4 0 66.7 93.3 100 82.1

Discussion

The immune response generated following immunization with formulations(Table 7) was assessed by measurement of anti influenza (A/PR8 strainspecific) response. Results are illustrated in FIGS. 7 and 8. Group(formulation) 3.1 which consisted of both HA DNA and proteinco-delivered in the same liposomal formulation produces a greaterresponse than all the other groups (formulations) at each sera samplebleed tested (day 21 and day 42 (day 14 following the second dose)). Theresponse for this co-delivered formulation group (3,1,1) is greater interms of magnitude of both OD450 nm sera dilution and titre (endpointread OD 0.3 units).

The response to the payload components, HA DNA and protein admixed(group 3.3), consistently generates a weaker immune response than thesame payload components co-delivered (group 3.1). Indeed, using the sameDNA payload (at 10 μg DNA dose) with a reduced protein payload (0.5 μgprotein dose) in the same liposomal vehicle (group 3.2), produces anequivalent serum Ig immune response to HA DNA and protein admixed with3-fold greater protein component payload (group 3.3). Group 3.4 failedto produce any specific anti Ig influenza response, however this groupreceived no immunogenic components (PBS only) thus this result is asexpected.

The live virus challenge results serve to indicate if the immuneresponse induced in the mice in response to immunisation with theformulations is adequate to protect the animals from virus infection.Group 3.4 serves as a negative control, as these are essentially ‘naive’animals they reflect the normal profile of the virus infection followingchallenge. In this group (3.4) all animals are infected with virus byday 4, with an average % (over 5 days) of 68% (sem 18%) animalsinfected. Group 5.3 which consisted of the pay load components, HA DNAand protein admixed, non liposomally co-delivered whilst inducing ananti influenza response FIG. 7 failed to demonstrate a significant(relative to group 3.4) reduction in the % of animals are infected withvirus with an average % (over 5 days) of 37% (sem 10% animals infected.Group (formulation) 3.1 which consisted of both HA DNA and proteinco-delivered in the same liposomal formulation produced a greaterresponse antibody response FIG. 7 and 8 than all the other groups(formulations) and demonstrates a significant (relative to group 3.4)(p<0.05) reduction in the % of animals are infected with virus with anaverage % (over 5 days) of only 7% (sum 6%) animals infected.

In summary we have found that the present invention is highly effectivefor generating an immune response, which is capable of protecting anindividual from infection with an infectious organism when administeredto a subject. The response involves an antibody response. Theimprovement exhibited by the present invention involves composition ofliposome forming materials delivering a payload of nucleic acidoperatively encoding an antigenic protein and an co-delivered protein,wherein the co delivered protein shares epitopes with the antigenicprotein.

Example 4 Multivalent Influenza Vaccine Materials and Methods Lipids

Egg phosphatidylcholine (PC), Dioleoyl phosphatidyl-ethanolamine (DOPE)and 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) were purchasedfrom Sigma Chemical Co,. UK. All lipids were stored (−20° C) dissolvedin chloroform, purged with nitrogen.

DNA

The plasmids pII7/HA-Sichuan and pI.18/PR 8 -HA were provided by Dr. J.Robertson (NIBSC, UK)) and contain the full length HA sequence from,respectively, Influenza A/Sichuan/2/87 and Influenza A/Puerto Rico/8/34.Plasmids for dosing were commercially produced by Aldevron (Fargo, USA)and contained <100 Endotoxin Unit (EU)/mg of DNA with no residualprotein delectable.

Proteins

Influenza A/Sichuan/2/87 and Influenza A/Puerto Rico/8/34 wholeinactivated virus proteins (sucrose gradient purified, major protein HA,ref antigen HA) were obtained from NIBSC, UK.

Preparation of Liposome Composition

Briefly, small unilamellar vesicles (SUV) were prepared front eggphosphatidylcholine (PC) and dioleoyl phosphatidylcholine (DOPE) and1,2-dioleoyloxy-3-(trimethyl-ammonium) propane (DOTAP) (4:2:1 molarratio) by sonication were mixed with either pI17/HA-Sichuan DNA andInfluenza A/Sichuan/2/87 virus protein or pI.18/PR8-HA DNA and InfluenzaA/Puerto Rico/8/34 virus protein (see) Table 10. Formulations wereprepared in duplicate, one vial for dosing and one vial for % entrapmentcalculations based radio labeled tracer (HA; DNA and protein) added toentrapped materials and freeze-dried overnight as described inGregoriadis, G., Saffie, R. and Hart, S. L., “High yield incorporationof plasmid DNA within liposome: effect on DNA integrity and transfectionefficiency,” J Drug Targeting (1996) 3(6):467-475 and in Kirby, C.,Gregoriadis, G., “Dehydration-rehydration vesicles (DRV): A new methodfor high yield drug entrapment in liposomes,” Biotechnology (1994)2:979-984. Following rehydration under controlled conditions, thegenerated dehydrated-rehydrated vesicles (DRV liposomes) were washed bycentrifugation to remove non-incorporated DNA. The washed pellets wereresuspended in PBS to the required dose volume. DNA and/or proteinincorporation into was estimated on the basis of ³⁵S (for DNA) and ¹²⁵I(for protein) radioactivity recovered in the suspended pellets.Liposomes were subjected to microelectrophoresis and photon correlationspectroscopy (PCS) at 25° C. in a Malvern Zetasizer 3000 to determinetheir zeta potential (ZP) and z-average diameter respectively.

Animal Procedure

Female Balb/c mice 6-12 weeks old (Harlan, UK) were immunised bysubcutaneous injection administered in 0.2 ml dose volume. Final dosequantities are summarised in Table 10. Mice received one dose on day 0,with sample bleeds collected from the tail vein at days 14 and 28. Theliposomal composition for group 4.3 was an admixture of the compositionsfor groups 4.1 and 4.2 mixed immediately before administration.

TABLE 10 Dose Quantity Total/animal Group pI17/HA- A/Sichuan/2/87pI.18/PR8- A/PuertoRico/8/34 (formulation) Sichuan DNA virus protein HADNA virus protein Lipid mg 4.1 10 μg 1.5 μg — — 2.1 4.2 — — 10 μg 1.5 μg2.1 4.3 10 μg 1.5 μg 10 μg 1.5 μg 4.2 4.4 — — — — nil (PBS)

Sera ELISA

Sera obtained from sample bleeds were diluted in PBS and kept at −20° C.until assayed by the enzyme-linked immunoadsorbent assay (ELISA). Twodifferent protocols were used depending of the protein substrate beingdetected.

For HA-PR8, certified binding chemistry 96-well plates were coated with50 μl/well of Influenza IIA-PR8 antigen (20 μg/ml) in PBS. Afterovernight incubation at 4° C. wells were washed four times withPBS/Tween 20™ (PBST) then wells were coated with 200 μl of 2% (w/v) BSAin PBS. After 1 h at 37° C., the blocking solution was removed and wellswere washed four times with PBST and overlaid with 50 μl/well ofdilutions of different experimental serum (individual animal samplebleeds) starting at dilution 1/100. Following 1 h incubation at 37° C.,plates were washed four time with PBST and overlaid 50 μl/well of rabbitanti-mouse Ig-HRP conjugated sera (Dako). After 1 h at 37° C., plateswere washed four tunes with PBST and overlaid with 50 μl/well ofsubstrate solution 3,3′5,5′ tetramethylbenzidine (TMB, Pierce). Thereaction was slopped by adding 50 μl/well of stopping solution (2Msulphuric acid) and the absorbance of each well at 450 nm wasdetermined.

For HA-Sichuan, certified binding chemistry 96-well plates were coatedwith 50 μl/well of a 1/2000 dilution of anti-HA Sichuan sheep serum(NIBSC standard reagent) in 0.1M Carbonate buffer (pH 9.5). Afterovernight incubation at 4° C. wells were washed four times withPBS/Tween 20™ (PBST) then wells wore dialed with 200 μl of 2% (w/v) BSAin PBS After 1 h 37° C., the blocking solution was removed and wellswere washed four times with PBST and overlaid with 50 μl/well ofHA-Sichuan antigen (5 μg/ml) in PBS. After 1 h at 37° C., the antigensolution was removed and wells were washed four times with PBST andoverlaid with 50 μl/well of dilutions of the different experimentalserum (individual animal sample bleeds) starting at dilution 1/100.Following 1 h incubation at 37° C., plates were washed four times withPBST and overlaid 50 μl/well of rabbit anti-mouse Ig-HRP conjugated sera(Dako). After 1 h at 37° C., plates were washed four times with PBST andoverlaid with 50 μl/well of substrate solution3,3′5,5′tetramethylbenzidine (TMB, Pierce). The reaction was stopped byadding 50 μl/well of stopping solution (2M sulphuric acid) and theabsorbance of each well at 450 nm was determined.

Results

The liposomal physical characteristics (% product (DNA and/or protein)entrapment, particle size and surface potential (Zeta)) are summarizedin Table 11.

TABLE 11 % entrapment Size Zeta Group DNA Protein nm mV 4.2 96.7 92.2676 48.4 4.1 93.9 87.4 816 42.7

The antibody responses (Sera ELISA) are expressed as the mean (n=5animals/group) OD signal±SEM at the Log₁₀ serum dilution assayed.Results are expressed in FIGS. 9a -d.

Discussion

The immune response generated following immunization with formulations(Table 10) was assessed by measurement of anti influenza HA-PR8 andHA-Sichuan strain specific antibody responses. Results are illustratedin FIGS. 9a -d.

At day 14, a clear antibody response to the Influenza antigens wasdetected in all experimental groups with the exception of formulation 2(PBS). Immunization with formulation 4,3, consisting of HA DNA andproteins for both Influenza Sichuan and Influenza Puerto Rico 8, inducedequivalent antibody titers, within standard error of the mean (SEM), toeach of the strains as those induced blowing immunisation withformulation 4.1 (Influenza Sichuan DNA and protein) or formulation 4.2(Influenza Puerto Rico 8).

At day 28, there was marked increase in the antibody response to theInfluenza antigen compared to day 14. In addition, immunization withformulation 3, consisting of HA DNA and proteins for both InfluenzaSichuan and Influenza Puerto Rico 8, again induced equivalent antibodytiters, within SEM, to the Influenza Sichuan strain to those inducedfollowing immunization with formulation 4.1 (Influenza Sichuan DNA andprotein). In contrast, immunisation with formulation 4.3, consisting ofHA DNA and proteins for both Influenza Sichuan and Influenza Puerto Rico8, induced antibody titers to the Influenza Puerto Rico 8 strain belowthose induced following immunization with formulation 4.2 (InfluenzaPuerto Rico 8) at two dilution points tested.

In summary, we have found that the present invention, when applied tothe delivery of multivalent (e.g., multi-strain), is highly effective ininducing antibody responses to the different strains present in theformulation. This antibody response develops quickly (antigen specificantibody titers are >1000 only 14 days after a single immunization) andit can be of the same level as that induced by the present inventionwhen delivering only DNA and protein components of one single strain(e.g., monovalent formulations). Even in occasions when immunizationwith a multivalent formulation may induce a lower antibody response toone of the strains compared to that induced by the equivalent monovalentformulation, the level of this response is again high (>1000 titer) andincreases with time. Therefore, the improvement caused by the presentinvention, and exemplified in this experiment, is the ability to inducea clear antibody response to several different antigenic strainsfollowing a single immunization with a formulation containing DNA andprotein antigens from all these strains.

Example 5 Entrapment Levels of DNA and Protein and Liposome Sizes

The general method of liposome formulation noted in Example 1 was usedto entrap hepatitis B surface antigen and plasmid DNA encoding thatantigen, at various levels of protein, shown in Table 12. The percentageentrapment values are shown in Table 12 and Table 13 shows the averagesize and zeta potential of the liposomes.

TABLE 12 Entrapment of HbsAg protein and/or DNA encoding HbsAg intoliposomes % Entrapment DNA Protein Lipid PC:DOPE:DOTAP DNA Protein 5.162.0 μg 17.68 μg 9.07:4.53:2.27 μM 98.9 64.4 6.96 + 3.37 + 1.59 mg(total lipid 11.92 mg) 5.2 62.0 μg 3.536 μg 9.07:4.53:2.27 μM 100 76.46.96 + 3.37 + 1.59 mg (total lipid 11.92 mg) 5.3 62.0 μg 0.697 μg9.07:4.53:2.27 μM 100 66.2 6.96 + 3.37 + 1.59 mg (total lipid 11.92 mg)5.4 — 17.68 μg 9.07:4.53:2.27 μM — 74.9 6.96 + 3.37 + 1.59 mg (totallipid 11.92 mg) 5.5 — 3.536 μg 9.07:4.53:2.27 μM — 80.4 6.96 + 3.37 +1.59 mg (total lipid 11.92 mg) 5.6 — 1.23 μg 9.07:4.53:2.27 μM — 93.86.96 + 3.37 + 1.59 mg (total lipid 11.92 mg)

Table 12 shows that entrapment of DNA was highly efficient, whereas thatof the HbsAg protein was moderately less so. The presence of plasmid DNAhad a modest negative effect on the efficiency of entrapment of protein.However, when protein and DNA were entrapped together, there was nonegative effect on the entrapment of DNA. These data demonstrate thatthe efficient co-entrapment of DNA and protein in these liposomalformulations is not unique to the influenza-A hemagglutinin, or uniqueto any one plasmid. It is likely that if efficient entrapment of DNA andprotein is a general property of these liposomal compositions,applicable to virtually any combination of plasmid DNA and proteinantigen.

TABLE 13 Z Average size (nm) & zeta potential (mV) for liposomes used inHbsAg formulations Formulation Size nm Zeta potential mV 5.1 547 +45 5.2710 +37 5.3 704 +39 5.4 666 +41 5.5 694 +33 5.6 639 −10

It is evident from these data on liposomal formulations of hepatitis-Bsurface antigen and various plasmid DNAs, that the size range isappropriate to uptake by classical antigen presenting cells such asmacrophages and dendritic cells. Uptake of materials from theseliposomes by B-cells is likely to require same degree of fragmentationor degradation in vivo or evolve liposomes of smaller size within theheterogeneous population of liposome formulation.

Example 6 Non-Phospholipidic Formulations Vehicle Materials

Materials 1-monopalmitoyl-rac-glycerol (C16:0) (Monopal), cholesterol(CHOL) and 3 beta-[N-(N′,N′-dimethylamino-ethane)carbamoyl] cholesterol(DC-Chol) were purchased from Sigma Chemical Co., UK. All materials werestored (−20° C.) dissolved in chloroform, purged with nitrogen.

DNA

Plasmid pCI-OVA (ret DNA OVA) (a kind gift of Dr. T. Nagata, HamamatsuUniversity School of Medicine, Japan) contains the chicken egg albuminprotein (ovalbumin, OVA) (Yoshida A, Nagata T, Uchijima M, Higashi T,Koide Y, “Advantage of gene gun-mediated over intramuscular inoculationof plasmid DNA vaccine in reproducible induction of specific immuneresponse,” Vaccine (2000) 18:1725-1729) cDNA cloned at the EcoR1 site ofthe pCI plasmid (Promega, Madison, Wis.) downstream from the CMVenhancer/promoter region. Plasmid p1.18/PR8-HA (ref DNA HA) was providedby Dr. J. Robertson (NIBSC, UK) containing the full length HA frominfluenza A/Puerto Rico/8/34. Plasmid for dosing was commerciallyproduced by Aldevron (Fargo, USA) and contained <100 Endotoxin Unit(EU)/mg of DNA with no residual protein detectable.

Proteins

Influenza A/Puerto Rico/8/34 whole inactivated virus protein (sucrosegradient purified, major protein HA, ref antigen HA) was obtained fromthe NIBSC UK.

Preparation of Compositions

Delivers system vehicles were prepared from Monopal and Chol and DC-Chol(4:2:1 molar ratio) by film drying under vacuum the resulting film washydrated with water by shaking for 1 h at 60° C., after cooling to RT,they were mixed with DNA (ref DNA HA or DNA OVA) and/or protein (refantigen HA) see Table 14. Formulations were prepared in quadruplicate,two vials for dosing (prime and boost) and two vial for % entrapmentcalculations based radio labeled tracer (DNA and protein) added toentrapped materials and freeze-dried overnight as described inGregoriadis, G., Saffie, R. and Hart, S. L., “High yield incorporationof plasmid DNA within liposome; effect on DNA integrity and transfectionefficiency,” J Drug Targeting (1996) 3(6):467-475 and in Kirby, C.,Gregoriadis, G., “Dehydration-rehydration vesicles (DRV): A new methodfor high yield drug entrapment in liposomes,” Biotechnology(1994)2:979-984. Following rehydration under controlled conditions, thegenerated dehydrated-rehydrated vesicles (DRV) were washed bycentrifugation to remove non-incorporated DNA. The washed pellets wereresuspended in PBS to the required dose volume. DNA and/or proteinincorporation into was estimated on the basis of ³⁵S (for DNA) and ¹²⁵I(for protein) radioactivity recovered in the suspended pellets. Vehicleswere subjected to microelectrophoresis and using laser diffraction at25° C. in a Malvern Zetasizer 3000 and Malvern Mastersizer to determinetheir potential (ZP) and z-average diameter respectively.

Animal Procedures

Female Balb/c mice 6-12 weeks old (Harlan, UK) were immunised bysubcutaneous injection administered in 0.2 ml dose volume. Final dosequantities are summarised in Table 14. Mice received two doses on days 0and 28, with sample bleeds collected from the tail vein at day 21 (post1 dose) and 42 (post 2 doses) with respect to the first injection.

TABLE 14 Group Dose Quantity Total/animal (formulation) DNA μg Protein(HA) μg Vehicle mg 6.1 10 (HA) 1.5 5.7 6.2 10 (OVA) 1.5 5.7 6.3 10 (HA)1.5 11.4 6.4 nil 1.5 5.7 6.5 10 nil 5.7 6.6 nil 1.5 nil (PBS)

In the composition of groups 6.1 and 6.2 the DNA and protein werecoentrapped. In the composition of groups 6.3 the DNA and protein wereseparately entrapped and admixed, i.e., this was an admixture of 6.4 and6.5. In group 6.6 the protein was not entrapped.

Sera ELISA

Sera obtained form sample bleeds were diluted in PBS and kept at −20° C.until assayed by the enzyme-linked immunoadsorbent assay (ELISA).Certified binding chemistry 96-well plates were coated overnight with 50μl/well of Influenza HA-PR8 antigen (20 μg/ml) in PBS. Incubateovernight at 4° C. After overnight incubation wells were washed fourtimes with PBS/Tween 2™ (PBST) then wells were coated with 200 μl of 2%(w/v) BSA in PBS. After 2 h at 37° C., the blocking solution was removedand wells were washed four times with PBST and overlaid with dilutionsof the different experimental serum (individual animal sample bleeds)starting at dilution 1/100 (50 μl sample/well). Allowing 1 h incubationat 37° C., plates were washed four times with PBST and overlaid 50μl/well of rabbit anti-mouse Ig-HRP conjugated sera (Dako). After 1 h at37° C., plates were washed four times with PBST and overlaid with 50μl/well of substrate solution 3,3′,5,5′ tetramethyl-benzidine (TMB,Pierce). The reaction was stopped by adding 50 μl/well of sloppingsolution (2M sulphuric acid) and the absorbance of each well at 450 nmwas determined.

Results

The vehicle physical characteristics (% product (DNA and/or protein)entrapment, particle size and surface potential (Zeta)) are summarizedin Table 15.

TABLE 15 Dose 1 Dose 2 Dose 1 Dose 2 % entrapment % entrapment Size ZetaSize Zeta Group DNA Protein DNA Protein nm mV nm mV 6.1 96.7 89.7 98.388.0 4140 23 3770 ND 6.2 94.6 90.2 79.4 82.8 4620 23.9 2950 ND 6.4 ND86.9 ND 92.8 4550 23.6 4060 ND 6.5 97.2 ND 97.2 ND 4150 24.9 3580 ND ND= not determined.

The individual animal (n=5/group) antibody responses (Sera ELISA) areexpressed as the reciprocal serum dilution required for OD to reach areading of 0.270 (end point dilution, ˜×2 normal mouse sera OD at 1/100dilution assayed). Results are expressed in FIG. 10.

Discussion

The immune response generated following immunization with formulations(Table 14) was assessed by measurement of anti influenza (A/PR8 strainspecific) response. Results are illustrated in FIG. 10. Group(formulation) 6.1 which consisted of both HA DNA and proteinco-delivered in the same delivery vehicle produces a greater responsethan all the other groups except group 6.2.

The results indicate that: delivery of HA protein oilers no advantageover protein alone (Group 6.4 vs Group 6.6), delivery of HA DNA aloneproduces no significant antibody response (Group 6.5) indeed 4 out of 5animals fall to induce an response greater than the limit of detectionof the assay (1/100 dilution sera), admix delivery of HA DNA and proteinin separate vehicles (Group 6.3) offers no advantage over protein aloneor vehicle delivered protein (Group 6.6 and 6.4 respectively) andco-delivery of HA Protein with a DNA (HA or OVA, Groups 6.1 and 6.2)component generates a significantly higher anti HA response than, admixdelivered material or material, delivered alone (Groups 6.3, 6.4. 6.5and 6.6 respectively).

In the context of this invention the last observation is applicable toboth the “cognate” and “irrelevant” DNA component. The immune systemresponse assayed are restricted to antibody responses and cellularmediated immune response (T helper, CTL etc) have not examined. Thusequivalence in immune responses to “cognate” and “irrelevant” DNA groups(Group 6.1 and 6.2) cannot be concluded. Indeed, HA DNA aloneimmunisation (Plasmid DNA encoding influenza virus haemagglutinininduces Th1 cells and protection against respiratory infection despiteits limited ability to generate antibody responses. Johnson P A, ConwayM A, Daly J. Nicolson C, Robertson J. Mills K H., J Gen Virol. (2000)Jul;81(Pt 7); 1737-1745, has been found to provide protection frominfluenza challenge in the absence of antibody responses thus basedcellular mediated immune response alone. As group 6.1 “cognate” codelivery posses HA DNA as a active component of the formulation andgroup 6.2 “irrelevant” does not contain HA DNA as a active component ofthe formulation, it does not seem unreasonable to suggest that group 6.1may induce an additional cellular mediated immune response which has notbeen measured.

In summary, we have found that the present invention is highly effectivefor generating an immune response. The response involves an antibodyresponse. The improvement exhibited by the present invention involvescomposition of a delivery vehicle without phospholipid componentsdelivering a payload of nucleic acid operatively encoding an antigenicprotein and a co delivered protein.

1. A process for forming a liposomal composition comprising: (a)providing an aqueous suspension of small unilamellar (SUVs) formed ofliposome forming materials; (b) contacting the aqueous suspension ofSUVs with nucleic acid which operatively encodes an antigenic protein toform an SUV-nucleic acid suspension; (c) dehydrating the SUV-nucleicacid suspension to provide a dehydrated mixture: and (d) rehydrating thedehydrated mixture in an aqueous resuspending medium to form asuspension of nucleic acid containing liposomes; Including the step ofintroducing an assistor protein whereby the nucleic acid containingliposomes are associated with said assistor protein.
 2. The processaccording to claim 1, wherein the assistor protein is contacted with theaqueous suspension of SUV's before, during or after step b and beforestep c.
 3. The process according to claim 1, wherein the assistorprotein is present in the resuspending medium.
 4. The process accordingto claim 1, wherein the liposome forming materials comprise at least onephospholipid and at least one sterol.
 5. The process according to claim1, wherein the materials comprise at least one cationic compound and theliposomes have an overall cationic charge.
 6. The process according toclaim 1, wherein the weight ratio of nucleic acid to liposome formingmaterials is in the range 1:100 to 1:1000.
 7. The process according toclaim 6, wherein the weight ratio of nucleic acid to liposome formingmaterials is in the range of 1:100 to 1:300.
 8. The process according toclaim 1, wherein the weight ratio of nucleic acid to assistor protein isin the range of 1000:1 to 1:1.
 9. The process according to claim 8,wherein the weight ratio of nucleic acid to assistor protein is in dierange of 30:1 to 5:1.
 10. The process according to claim 1, wherein thedehydration is by freeze-drying.
 11. The process according to claim 1,wherein step (b) further comprises contacting the aqueous suspension ofSUVs with a second nucleic acid which operatively encodes a secondantigenic protein; and the step of introducing an assistor proteinfurther comprises introducing a second assistor protein whereby thenucleic acid containing liposomes are associated with said secondassistor protein.